Transfer vector

Abstract

A recombinant DNA vector is provided as a universal transcription vector having a replication origin and selectable marker, a promoter and a transcription initiation site comprising a first transcribed nucleotide, wherein a restriction site is provided immediately adjacent to and upstream from the transcription initiation site so as to seprate transcribed from untranscribed nucleotides. A second restriction site may also be positioned downstream from the said restriction site. Precise control of initiation and termination of transcription is attained by this invention. Such control is important in assuring the effectiveness of transcribed RNA viral vectors. A high fidelity in vitro RNA transcription method is also provided utilizing vectors constructed from the universal transcription vector, or other vectors producing transcripts having no more than one extra 5' base. This method is capable of producing functional RNA transcripts, preferably comprising infectious viral sequences.

What is claimed is: 
     
       1. An in vitro process for preparing high tolerance RNA transcripts, said process comprising: (a) preparing a cDNA sequence corresponding to the RNA transcript desired;   (b) providing a promoter sequence attached to said cDNA sequence, said promoter sequence directing transcription of a first transcribed nucleotide wherein said first transcribed nucleotide is the first nucleotide of said cDNA sequence or is immediately 5' to said first nucleotide of said cDNA sequence;   (c) replicating said cDNA sequence and attached promoter sequence;   (d) placing replicated sequences of step (c) in an in vitro environment comprising an RNA polymerase regulated by said promoter sequences so as to permit transcription of RNA transcripts corresponding to said cDNA;   (e) isolating RNA transcripts from the materials of step (d).   
     
     
       2. The process of claim 1 wherein said high tolerance RNA transcripts are selected from the group of RNA sequences consisting of infectious viral sequences, tRNA, mutated tRNA, rRNA, mutated rRNA, snRNA and mutated snRNA. 
     
     
       3. The process of claim 1 wherein said RNA transcripts are capable of translation into proteins. 
     
     
       4. The process of claim 1 wherein said RNA transcripts comprise infectious viral sequences. 
     
     
       5. The process of claim 4 wherein said RNA transcripts also comprise RNA heterologous to the naturally-occurring RNA from which the cDNA sequence was prepared. 
     
     
       6. The process of claim 4 wherein said RNA transcripts comprise symptomless viral sequences. 
     
     
       7. The process of claim 1 wherein said promoter sequences are selected from the group consisting of PR lambda phage, T7, T3, SP6 and synthetic promoter sequences. 
     
     
       8. The process of claim 7 wherein said promoter sequences comprise PR lambda phage promoter sequences. 
     
     
       9. The process of claim 7 wherein said promoter sequences comprise T7 promoter sequences. 
     
     
       10. The process of claim 7 wherein said promoter sequences comprise SP6 promoter sequences. 
     
     
       11. The process of claim 7 wherein said promoter sequences comprise T3 promoter sequences.